Biodegradable microspheres as a carrier for macromolecules

ABSTRACT

A method for preparing biodegradable microspheres having a three-dimensional network in which biologically active macromolecular agents are physically entrapped therein. The microsphere is able to degrade and release the macromolecular agent at a controlled rate. The method involves emulsifying a vinyl derivative of a biodegradable hydrophilic polymer, a water-soluble monovinyl monomer and a biologically active macromolecule in water, and copolymerizing the biodegradable hydrophilic polymer and the water-soluble monovinyl monomer such that the biologically active macromolecule is entrapped therein.

This application is a continuation of application Ser. No. 07/152,843,filed Feb. 5, 1988, now abandoned, which is a divisional of applicationSer. No. 06/864,147, filed May 16, 1986, now U.S. Pat. No. 4,741,872.

BACKGROUND OF THE INVENTION

(1) Field of the Invention

The present invention relates generally to the field of biodegradablepolymers for the controlled release of biologically active agentstherefrom. More particularly, the present invention relates to a processfor preparing biodegradable polymers in the form of spherical particlesof controlled size. The process is designed to allow the biodegradablepolymer particles to contain incorporated biologically active agents andto allow controlled release of these agents while allowing targeteddelivery via injection or inhalation.

(2) Background of the Prior Art

The use of proteins and peptides as therapeutic agents has beenrecognized and their position within the pharmaceutical armamentarium isgrowing due to their increasing availability. This availability isprimarily due to recent advances in genetic engineering andbiotechnology. Unfortunately, the use of proteinaceous drugs byconventional routes of administration is generally hampered by a varietyof delivery problems. Nonparenteral routes of administration, i.e., oraland percutaneous, are inefficient primarily due to poor absorption ofproteinaceous drugs into the bloodstream and degradation of such drugsin the gastrointestinal tract. Rapid proteolytic inactivation of theproteinaceous drug also occurs when the drug is administeredparenterally thus decreasing its bioavailability. In addition, whenadministered by the parenteral route, the host's immune system isactivated thereby potentially setting off a series of undesirable immunereactions.

In view of the foregoing, considerable effort has been devoted todeveloping alternative systems for parenteral delivery of peptides andproteins to obviate the problems associated with prior artadministration techniques. For instance, implantable devices have beencast or molded from poly-(hydroxyethyl)methacrylate, polyvinyl alcohol,ethylene-vinylacetate copolymer (EVA) and silicone elastomer.Macromolecular drugs have been embedded in those devices. A typicalmethod of preparation involves suspending a powder of a macromoleculardrug such as a solid protein or peptide in a solution containing thepolymer. The entire composition is then cast or molded into the desiredsize and shape either by evaporating the solvent or by vulcanization. Asustained release of macromolecules from these devices has beendemonstrated. The simplicity of the foregoing prior art method is itsprimary advantage.

However, one disadvantage of hydrophobic polymers such as those preparedfrom EVA and silicon, is that those polymers are not permeable tohydrophilic macromolecules, thus, only that portion of the drug whichcommunicates with the surface of the implant, either directly or viacontact with other drug particles, can be released. Thus, the drugpresent nearer the interior of the implant and completely surrounded bythe polymer matrix is unable to ever be released and never exerts itstherapeutic effect. Addition of polar additives increases penetration ofwater in these hydrophobic materials and helps to dissolve the protein,but they are not quite inert to the protein, as are the polar organicsolvents used for casting from PHEMA and PVA. Another disadvantageassociated with these types of devices is the need for surgicalinsertion and eventually surgical removal of the implant. This isnecessary since the devices are composed of materials which arenondegradable.

Microspheres containing proteins have been prepared from polyacrylamide,acryloylated dextran and acryloylated starch. Polyacrylamide beads canmeet different purposes in vitro, but their nondegradability preventstheir use in humans. Reported data on polysaccharide particles show thatan efficient crosslinking has been achieved only at a high degree ofderivatization (D.D. about 0.1 to 0.3). A high D.D. is disadvantageousas it decreases the biocompatibility of the polymer. A high D.D. alsoleads preferentially to the intramolecular reaction of polymerizablegroups instead of the intermolecular reaction between different polymerchains, which results in a heterogenous microporous structure. The useof the crosslinking agent bisacrylamide is not considered desirable,since it generally results in the formation of crosslinked hydrocarbongels, which neither dissolve nor degrade even after degradation of thepolysaccharide component.

The recent advances in the incorporation of drugs into microparticulatecarriers has attracted a great deal of attention because it combinesfeatures of matrix-controlled release with those of injectable forms. Inaddition to controlled release, these microspherical carriers offer"first stage" physical targeting, that is, physical localization of thedrug carrier in the proximity of the target tissue and cells. Localizedadministration of the therapeutic agent allows for not only moreefficient drug therapy but also minimizes the opportunity for adversesystemic effects.

In preparing microspheres in the size range of 1 μm to 20 μm, homogenoussystems are more suitable than heterogenous systems for castingimplants. In the homogenous system, proteins are co-dissolved in thesame solvent as the material of the matrix. Furthermore, in order topreserve the biological activity of the macromolecules, aqueous systemsare generally preferred. In this regard, biodegradable hydrophilicpolymers can be chosen as matrix material provided that they can besolidified or crosslinked by a mechanism which does not involve achemical modification and/or denaturation of the incorporatedmacromolecule such as a proteinaceous agent.

It is known that crosslinked hydrophilic gels can be obtained utilizingtechniques of free-radical polymerization. To some extent, the problemsidentified above are similar to those found in the preparation of graftbiodegradable polymers, that is, polymers containing vinylic groups withthe encapsulation of biologically active materials therein.

Examples of prior art patents include U.S. Pat. Nos. 4,131,576,3,687,878, 3,630,955 and 3,950,282. These patents disclose methods forthe preparation of graft copolymers of polysaccharides and vinylicmonomers. These patents were directed to improving the physicalproperties of the polysaccharides within each composition. Processconditions used to achieve these improvements included the use ofelevated temperatures, highly reactive monomers or organic solvents.However, each of the foregoing parameters are harmful to biologicallyactive macromolecules and thus are unsuitable in the practice of thepresent invention.

The prior art also discloses procedures for encapsulation of a corematerial in a polymer capsule. U.S. Pat. No. 4,382,813 discloses theproduction of a capsule wall by the gelation of polysaccharide gums,such as alkali-metal alginates, with bivalent metal cations. U.S. Pat.No. 4,344,857 discloses the gelation of xanthates of polyhydroxypolymers by the addition of strong acids and coupling agents. U.S. Pat.No. 3,567,650 achieves a similar result by lessening the solubility ofcertain polymeric materials using increasing temperature.

Other mechanisms are based on the principle of complex coacervationusing at least two colloids of opposite electrical charge and oxidationproducts of polysaccharides as crosslinking agents as disclosed in U.S.Pat. No. 4,016,098. Yet another procedure employs interfacialcrosslinking of the wall-forming polymer by reactive bifunctionalcrosslinking agents dissolved in oil droplets which are encapsulated astaught in U.S. Pat. No. 4,308,165. Other examples of the prior art whichoffer similar teachings include U.S. Pat. Nos. 4,078,051, 4,080,439,4,025,455 and 4,273,672. Materials which are encapsulated according tothe prior art are mostly water insoluble solids or oil droplets andcompounds dissolved therein, e.g., dyes, pigments or biologically activelow-molecular-weight compounds like herbicides.

U.S. Pat. No. 4,352,883 teaches a method for encapsulation of corematerials such as living tissues or individual cells in a semipermeablemembrane. The membrane is permeable for small molecules but notpermeable to large molecules. This patent also utilizes the gelation ofcertain water-soluble gums by the action of multivalent cations.

U.S. Pat. No. 4,038,140 discloses the procedure for binding ofbiologically active proteins onto an insoluble carrier by reacting theproteins in an aqueous phase with a carrier comprising an activatedpolysaccharide having a hydrophilic graft copolymer incorporatedtherein. That patent is directed to the preparation of insolublecarriers containing covalently bound proteins with application inbiochemical reactors.

Yet another example of the prior art, U.S. Pat. No. 4,094,833, teaches aprocedure for preparation of copolymerizates of vinylic group containingdextran and divinyl compounds, optionally also monovinyl compounds, inthe form of three-dimensional gel networks. The resulting crosslinkeddextran-vinylic gels can be used for separation purposes.

In spite of the numerous teachings of the prior art, the prior art doesnot offer a method for obtaining encapsulated or entrapped biologicallyactive macromolecules such as proteinaceous agents in sphericalmicroparticles of controlled size ranges. Nor does the prior art suggesta procedure for allowing microspheres to have the potential to controlthe rate by which the biologically active macromolecule is released orfor modulating the rate by which the matrix is degraded in vivo.

SUMMARY OF THE INVENTION

It is, therefore, the object of this invention to provide a process forthe incorporation of sensitive biologically active macromolecules,preferably peptides and proteins, into a biodegradable and biocompatiblematrix under conditions sufficiently mild to retain the biologicalactivity of the incorporated macromolecular agents.

It is another object of this invention to provide for a matrix,containing pharmacologically active macromolecules, in the form ofspherical particles of controlled size, preferably having a diameter inthe range of about 0.5 μm to about 500 μm.

It is also an object of this invention to produce microsphericalcarriers from which macromolecular agents are released under in-vivoconditions at a predictable rate.

It is yet another object of this invention to produce microsphericalcarriers of biologically active macromolecules which possess a potentialfor controlling the rate of biodegradation of the matrix so that therelease of the macromolecular agents can be regulated by thebiodegradation of the matrix.

A further object of the present invention is to produce microsphericalcarriers of biologically active macromolecules which possess a potentialfor controlling the rate of biodegradation of the matrix by adjustingthe matrix properties thereby controlling both release of themacromolecular agent and existence of the matrix in the tissue as wellas assuring the biodegradation of the matrix into nontoxic solubleproducts which are metabolized and/or excreted.

A still further object of the present invention is to provide amicrospherical drug delivery system which allows targeting of drugs orother agents to specific host tissues or cells via injection orinhalation providing high localized concentrations, sustained activity,systemic administration and treatment, thereby minimizing undesirablesystemic effects of toxic drugs administered directly into thecirculation.

These and similar objects, advantages and features are accomplishedaccording to the methods and compositions of the following descriptionof the present invention.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts an overall scheme for preparation of the biodegradablemicrospheres of the present invention.

FIG. 2 represents a more detailed view of the microsphere prepared bythe process depicted in FIG. 1.

FIG. 3 depicts the cumulative release of alpha-1-proteinase inhibitorfrom hydroxyethyl starchpolyacrylamide microspheres in μg of protein permg of microspheres.

DESCRIPTION OF THE PREFERRED EMBODIMENTS

The present invention provides a process for the incorporation ofsensitive biologically active macromolecules into a biodegradablematrix. The biodegradable matrix is prepared by the copolymerization ofa vinyl derivative of biodegradable hydrophilic polymer containing atleast two vinyl groups per polymer chain with a monovinyl water-solublemonomer. The biodegradable matrix is a three-dimensional gel network inwhich biologically active macromolecules are physically entrapped. Thebiodegradable matrix is particularly well-suited for the parenteralroute of administration.

According to the present invention, the biodegradable hydrophilicpolymer component of the matrix can be selected from a variety ofsources including polysaccharides, proteinaceous polymers, solublederivatives of polysaccharides, soluble derivatives of proteinaceouspolymers, polypeptides, polyesters, polyorthoesters, and the like.

The polysaccharides may be poly-1,4-glucans, e.g., starch glycogen,amylose and amylopectin, and the like. Preferably, the biodegradablehydrophilic polymer is a water-soluble derivative of a poly-1,4-glucan,including hydrolyzed amylopectin, hydroxyalkyl derivatives of hydrolyzedamylopectin such as hydroxyethyl starch (HES), hydroxyethyl amylase,dialdehyde starch, and the like.

Proteinaceous polymers and their soluble derivatives include gelationbiodegradable synthetic polypeptides, elastin, alkylated collagen,alkylated elastin, and the like.

Biodegradable synthetic polypeptides includepoly-(N-hydroxyalkyl)-L-asparagine, poly-(N-hydroxyalkyl)-L-glutamine,copolymers of N-hydroxyalkyl-L-asparagine and N-hydroxyalkyl-L-glutaminewith other amino acids. Suggested amino acids include L-alanine,L-lysine, L-phenylalanine, L-leucine, L-valine, L-tyrosine, and thelike.

Definitions or further description of any of the foregoing terminologyare well known in the art and may be found by referring to any standardbiochemistry reference text such as "Biochemistry" by Albert L.Lehninger, Worth Publishers, Inc. and "Biochemistry" by Lubert Stryer,W. H. Freeman and Company, both of which are hereby incorporated byreference.

The aforementioned biodegradable hydrophilic polymers are particularlysuited for the methods and compositions of the present invention byreason of their characteristically low human toxicity and virtuallycomplete biodegradability. Of course, it will be understood that theparticular polymer utilized is not critical and a variety ofbiodegradable hydrophilic polymers may be utilized as a consequence ofthe novel processing methods of the invention.

The three dimensional network or gel matrix according to the presentinvention is obtained by the free-radical polymerization of thebiodegradable hydrophilic polymer containing at least two vinyl orsubstituted vinyl groups with an additional monovinylic monomer.

The vinyl derivatives of the biodegradable hydrophilic polymer includederivatives containing groups of the formula (I):

    CH.sub.2 ═CR.sub.1 --(CH.sub.2).sub.n --X              (I)

wherein R₁ is a hydrogen atom or methyl group; n is 0, 1 or 2; and X isa radical having the formula ##STR1## wherein R₂ represents theabove-mentioned biodegradable polymer which contains at least two vinylor substituted vinyl groups per average polymer chain. Thus, Xrepresents an ether, secondary amine, ester or amide bridge between thegroup of formula (I) and the biodegradable hydrophilic polymer.Therefore, typical examples of vinyl substituents include vinyl, allyl,acryloyl, methacryloyl, acrylamido and methacrylamido groups.

The vinyl derivatives of the biodegradable hydrophilic polymer can beprepared in a variety of ways well known in the prior art. One suggestedapproach is the preparation of vinyl and allyl ethers by the reaction ofvinyl alkylhalides, allylhalides, vinylglycidyl ethers or allylglycidylethers with alkaline solutions of the selected biodegradable hydrophilicpolymer containing either hydroxyl or amino groups. In a like manner,derivatives containing either ester or amide linkages can be prepared byreacting acryloyl chlorides, methacryloyl chlorides, acryloyl glycidylesters or methacryloyl glycidyl esters with hydroxyl or amino groups ofthe biodegradable hydrophilic polymer.

The degree of derivatization (DD) of the biodegradable hydrophilicpolymer by the vinyl groups is such, that they are at least two vinylgroups per average polymer chain, preferably, at least three vinylgroups per average polymer chain. The upper limit of DD is given by thedesired density of crosslinking as discussed below. It should also benoted that the minimum DD, when expressed in moles of vinyl groups permole of monomer units of biodegradable hydrophilic polymer also dependson the molecular weight of the biodegradable hydrophilic polymer.

The monovinyl monomer has two functions. First, it is intended tofacilitate the propagation reaction of the growing radical by lesseningsteric hindrance during the polymerization of the macromolecular vinylderivatives. This obviates the necessity of a high degree ofderivatization of the starting biodegradable hydrophilic polymer. Andsecond, it is intended to introduce into the gel structure or matrix anondegradable component which can participate in the regulation of thedegradation rate of the matrix.

The ratio of the monofunctional monomer propagator to derivatizedbiodegradable hydrophilic polymer is chosen such, that during thepolymerization, short linear chains of hydrocarbon polymers are producedwhich are in fact crosslinked by degradable hydrophilic polymer chains.This assures that substantially the entire matrix of microspheres can bedegraded in vivo to low molecular weight soluble products.

The ratio between the biodegradable hydrophilic polymer component to thevinyl monomer component may be in the range of about 1:5 up to about40:1 based on a weight basis. Preferably, the ratio is in the range ofabout 2:1 to about 20:1.

The monovinyl monomer is designed to facilitate the propagation reactionof the growing radical during polymerization thereby obviating thenecessity of high derivatization of starting polysaccharide withpolymerizable groups. The monovinyl monomer also introduces in thepolymer matrix other functional groups, e.g., negatively or positivelycharged, which can participate in the control of drug release. Typicalfunctional groups which may participate in the control of drug releaseinclude carboxyl, amino, dialkylamino, dihydroxyalkylamino, and thelike. The presence of these positive or negative charges provideion-exchange properties to the matrix.

The monovinyl monomer may be selected from the group of hydrophilicesters and/or amides of acrylic or methacrylic acids, water-solublevinyl derivatives, acrylic acid, methacrylic acid, and the like. Typicalexamples of hydrophilic esters and/or amides of acrylic or methacrylicacids include acrylamide, methacrylamide, 2-hydroxyethyl methacrylate,2-hydroxypropyl methacrylamide,N-methylacryloyl-trishydroxymethylaminomethane,N-acryloyl-N'-dimethylaminopropylamine,3-N,N-dimethylaminopropylmethacrylamide, N-alkylmethacrylamide glycerylmonomethacrylate, and the like. Suitable water-soluble vinyl derivativesinclude N-vinylpyrrolidone, N-vinylimidazole, p-vinylbenzoic acid,vinylpyridine, and the like.

Suitable biologically active macromolecules intended to be used in thepractice of the present invention include hormones, proteins, peptides,vaccines, enzymes, enzyme inhibitors and other biologically activemacromolecules. A suggested inhibitor is alpha-1-antitrypsin (ATT), anα-proteinase inhibitor. Additional examples include amino acidmetabolizing enzymes in the treatment of neoplasia, fibrinolyticenzymes, interferon, growth hormone, antigens for desensitization,immunoglobulins and F_(ab) -fragments of immunoglobulins. The presentinvention is not intended to be limited to any of the foregoing andother types of biologically active macromolecules are equally suitablein the practice of the present invention.

The biologically active macromolecules remain free within the polymermatrix, that is, there are no chemical bonds between the macromoleculeor some other group within the microsphere. Thus, the macromolecule doesnot require the breakage of a chemical bond to be released. Releaseoccurs through diffusion out of the microsphere or biodegradable erosionof the polymer.

The polymerization reaction according to the present invention isconducted under suitable conditions for free radical polymerization. Thereaction is always conducted in aqueous solution. Suitable free radicalinitiators are redox type initiators. The polymerization reaction ispreferably conducted using free radical initiators to produce freeradicals under mild conditions such as a temperature of approximately 0°C. However, the temperature of the polymerization reaction may rangefrom about 0° C. to about 50° C. The preferred temperature at which toconduct the polymerization reaction ranges from about 0° C. to about 30°C.

It is a particularly advantageous feature of the present manufacturingprocedure that, starting from the dissolution of the macromolecule ofinterest until dispensing the final microspheres in vials, the entireprocess can be carried out at temperatures near 0° C. in order tominimize the denaturation effect on the macromolecule. Typical redoxtype initiators include ammonium persulfate, hydrogen peroxide, benzoylperoxide, and the like.

It is also advantageous to use a free radical initiator along with acompound which forms with the initiator a redox system and acceleratesthe formation of radicals. Examples of the second compound of theinitiator system include N,N,N'N'-tetramethylethylenediamine, ascorbicacid, N,N-dimethylamino-p-toluidine, 3-dimethylaminopropionitrile,sodium metabisulfite, and the like.

During the polymerization reaction, linear chains of vinylic polymer areformed which are cross-linked with the biodegradable hydrophilicpolymer. It is thus important that a monovinyl monomer is used duringthe polymerization reaction to ensure that only linear chains ofnondegradable hydrocarbon polymers are formed. Thus, the use of themonovinyl monomer ensures that the degradation of the biodegradablecomponent which is responsible for the crosslinking will allow for theformation of totally soluble degradation products. The monovinyl monomerof the present invention, since it is only a monomer, will have a lowmolecular weight compared to the biodegradable polymer. It has beenspeculated that if the molecular weight of the monomer exceeds 400, thensteric hindrance is possible. Thus, it is recommended for purposes ofthe present invention that the monovinyl monomer have a molecular weightof less than 400.

The drug delivery system in accordance with the present invention isideally suited for administration by parenteral or inhalation routes. Itwill be appreciated by those skilled in the art that the porousmicrospheres of the present invention containing incorporated drugs forrelease to target cells or tissues, therefore, may be administered aloneor in admixture with appropriate pharmaceutical diluents, carriers,excipients or adjuvants suitably selected with respect to the intendedroute of administration and conventional pharmaceutical practices. Theseinert pharmaceutically acceptable adjuvants are well known in the art.For example, for parenteral injection, dosage unit forms may be utilizedto accomplish intravenous, intramuscular or subcutaneous administration,and for such parenteral administration, suitable sterile aqueous ornon-aqueous solutions or suspensions, optionally containing appropriatesolutes to effect isotonicity, will be employed. Likewise for inhalationdosage forms, for administration through the mucous membranes of thenose and throat or bronchio-pulmonary tissues, suitable aerosol or sprayinhalation compositions and devices will be utilized.

The foregoing methodology allows for the preparation of microspheres incontrolled size ranges under conditions sufficiently mild to preservethe biological activity of functional macromolecules. In addition, theforegoing methodology allows for the potential for controlling therelease of the drug by controlling the crosslinking density and the rateof degradation via selecting the derivatization degree of the startingpolysaccharide and matrix composition.

The polymerization may be conducted by any polymerization process knownin the art, however, another important feature of the present inventionis the fact that the polymerization can be conducted using a beadpolymerization technique. According to the convenient process describedin the present invention, the derivatized biodegradable hydrophilicpolymer, the monovinyl monomer and the biologically active macromoleculewhich is to be incorporated therein are codissolved in an aqueous bufferof appropriate pH and ionic strength which is suitable for preservingthe biological activity of the macromolecular agent, usually togetherwith one component of the initiator system. Either oxidative orreductive types of initiators are useful.

The aqueous solution is then deoxygenated by purging with N₂ andemulsified in a deoxygenated water-immiscible organic liquid,preferentially composed of higher aliphatic hydrocarbons such as hexane,heptane, octane, cyclohexane, or their higher homologs and theirmixtures. In order to facilitate the emulsification and formation of awater-in-oil emulsion, appropriate emulsifying agents are added to thecontinuous organic phase. Typical emulsifying agents include sorbitanoleates, polyethylene glycol ethers, polyoxyethylene sorbitan esters,polyoxyethylene polyoxypropylene alcohols, and the like.

After obtaining an emulsion having a suitable size range of aqueousdroplets, the polymerization is begun by addition of the other componentof the initiator system to the emulsion. When a water soluble compoundis used, the oxidant component of the initiator system, e.g., ammoniumpersulfate and the like, is in the aqueous dispersed phase, then thesecond component is a reductant soluble in the continuous phase, e.g.,N,N,N',N'-tetramethylethylenediamine and the like. The microspheresformed by the polymerization of the aqueous droplets of the emulsion arecleansed by decantation and washed with an appropriate water-immiscibleorganic solvent and then freeze dried. Suitable organic water-immisciblesolvents include cyclohexane, benzene, cyclohexanone, and the like.

Following another procedure according to the present invention, themicrospheres after washing with organic solvent can be redispersed inwater or an aqueous buffer, washed with the buffer and freeze-dried froman aqueous suspension. The biologically active compound, e.g., peptide,protein, and the like, while co-dissolved in the aqueous dispersedphase, is entrapped in the crosslinked polymer network duringpolymerization and can be released in vivo essentially by the diffusionthrough the polymer network or following the degradation of the matrix.

A particularly advantageous feature of the foregoing process, andirrespective of the particular polymerization technique selected, isthat the microspheres can be prepared in a variety of size rangesgenerally ranging from about 0.5 μm to about 500 μm in diameter. Sizeranges from about 1.0 μm to about 15.0 μm in diameter are generallypreferred. For inhalation administration a microsphere size range offrom about 1.0 μm to about 5.0 μm in diameter is preferred. Forinjectable administration a microsphere size range of about 8.0 μm toabout 15.0 μm in diameter is preferred.

The size of the resulting microspheres depends on the size of theaqueous droplets in the water-in-oil emulsion. The size of the dropletsin turn is dependent upon the shear stress which is applied by thestirrer. The stirrer opposes the coalescing tendencies caused by surfacetension. Generally, the size of the droplets is reduced by applying ahigher shear stress. A higher shear stress is achieved either by using ahigher stirrer speed or by increasing the ratio between the viscositiesof the continuous phase and the dispersed phase. A higher viscosity ofthe continuous phase may be achieved by increasing the proportion ofhydrocarbons with more carbon atoms in the emulsion, e.g., octane,dioxane, dodecane and the like. The viscosity of the aqueous dispersedphase may be adjusted by using a different molecular weight of thestarting biodegradable hydrophilic polymer. Adjustment of the viscosityof the aqueous dispersed phase in this manner allows for use of the sametotal gel matrix and monovinyl monomer concentration.

Another advantageous feature of the present invention is the fact thatthe incorporated macromolecular agents are released from the gel matrixby a diffusion through the crosslinked hydrogel network. Various ratesof release of the macromolecular agents may be achieved by varying thecrosslinking density of the gel matrix. The crosslinking density of thematrix may be varied by selecting a biodegradable hydrophilic polymerwith varying degrees of derivatization (DD). Degrees of derivatizationare used to indicate the average distance between the attached vinylicgroups. A suitable crosslinking density is also dependent on themolecular weight of the macromolecular agent and on the desired rate ofits release.

The degree of derivatization is preferably in the range of about 0.01 toabout 0.20 mole of vinyl groups per mole of monomer units of thebiodegradable hydrophilic starting polymer. Preferably, there are about0.02 to about 0.15 mole of vinyl groups per mole of monomer units of thestarting polymer. If hydroxyethyl starch (HES) is used as the startingbiodegradable hydrophilic polymer, the broad range of about 0.01 toabout 0.20 corresponds to a molecular weight of the average segmentbetween crosslinking points of about 20,000 to about 1000, respectively.About 0.02 to about 0.15 corresponds to a molecular weight range of theaverage segment between the crosslinking points of about 10,000 to about1,800. The range in cross-linking density of 0.02 to 0.15 moles of vinylgroups per moles of monomer units will produce approximately a ten-folddifference in the release rate of the protein having a molecular weightof about 50,000.

It will be appreciated that the concentrations, temperatures and ratiosreferred to hereinabove and in the examples set forth operable rangesand that other numerical expressions may apply as different solvents,polymers, monomers, macromolecules, etc. are selected.

The following non-limiting examples are offered in order that thoseskilled in the art may more readily understand the present invention andthe specific preferred embodiments thereof. Unless indicated otherwise,all amounts are given in grams.

EXAMPLE 1

To a solution of hydroxyethyl starch (HES) (HESPAN, a trademark ofAmerican Critical Care) in dry, distilled N,N'-dimethylacetamide (DMAA)at approximately 0° C., a measured amount of distilled acryloyl chloridewas added in small portions along with an equimolar amount oftriethylamine, over approximately a 30 minute time period. The reactionvessel was maintained at this temperature and the reaction proceeded forapproximately 2 additional hours. The reaction mixture was thentransferred to a vessel containing 200 ml of acetone at about 0° C. toabout 5° C. to precipitate the polymer. The polymer was washed withacetone, dried with air suction, dissolved in water and reprecipitatedin acetone. Derivatized HES (acryloyl-HES) was finally purified bypreparative gel permeation chromatography in water and then freezedried.Ratios of the reactants and the data on the resulting polymers arepresented in Table 1. The symbol mw_(A) represents the molecular weightequivalent of the biodegradable hydrophilic polymer per vinyl group.D.D. represents the degree of derivatization in millimole/gram.

                  TABLE I                                                         ______________________________________                                        Preparation of Acryloyl-HES                                                              1a     1b       1c       1d                                        ______________________________________                                        HES          5.0      5.0      5.0    5.0                                     DMAA         18.8     18.8     18.8   18.8                                    Acryloyl chloride                                                                          0.1      0.2      0.4    1.0                                     Triethylamine                                                                               0.11     0.22     0.45  1.1                                     Acryloyl-HES (yield)                                                                       4.3      4.3      4.6    5.1                                     D.D. (mmole/gram)                                                                           0.07     0.17     0.26   0.62                                   mw.sub.A     14,300   6,000    3,800  1,600                                   ______________________________________                                    

EXAMPLE 2

Approximately 4.05 grams of partially hydrolyzed amylopectin wasdissolved in 80 ml of water. The solution was cooled to 0° C. and thesolution of 1.8 grams of acryloyl chloride in 10 ml of acetone was addedin small portions during stirring along with 10 ml of 2 N solution ofNaOH so that the solution was remained alkaline. After approximately 30minutes the acryloyl-amylopectin was precipitated with acetone andfurther processed in a manner similar to Example 1. The yield was 3.9grams and the D.D. was 0.32 mmole/gram.

EXAMPLE 3

Approximately 5.0 grams of HES was dissolved in 18.8 grams of DMAA andto this solution was added 6 ml of 2N solution of NaOH, 50 mg of4-methoxyphenol and 1.4 grams of allylglycidyl ether. The resultingmixture was stirred for 20 hours at room temperature and then processedin a manner similar to Example 1. The yield was 4.3 grams and the D.D.was 0.42 mmole/gram.

EXAMPLE 4

Approximately 4.3 grams of poly-[N-(2-hydroxyethyl)-L-glutamine],(PHEG), in 18.8 grams of DMAA was reacted with 0.4 grams of acryloylchloride in a procedure similar to that used in Example 1. The yield ofacryloyl-PHEG was 4.2 grams and the D.D. was 0.32 mmole/gram.

EXAMPLE 5

Acryloyl-HES, prepared according to Example 1, acrylamide andalpha-1-proteinase inhibitor (alpha-1-PI) were dissolved in 0.05mole/liter ammonium carbonate buffer pH 7.4, together with ammoniumpersulphate (2% mole/mole in terms of the total concentration of vinylgroups). The solution was deoxygenated by repeated evacuation andfilling of the vessel with nitrogen at 0° C. The deoxygenated solutionwas filtered and the filtrate was transferred to a polymerizationreactor containing 60 ml of organic continuous phase. The organiccontinuous phase was composed of a mixture of heptane, USP, mineral oiland 0.3 gram of SO-15 (sorbitan oleate). The entire mixture was thenflushed with nitrogen at 0° C. Table II provides a description of thecompositions of the dispersed and continuous phases. In Table II,average diameter (μm) represents the average diameter of themicrospheres after rehydration in 0.15 mole/liter NaCl and 0.05mole/liter phosphate pH 7.4. The protein content % represents thecontent of the diffusion releasable protein in dry microspheres.

The polymerization reactor consisted of a jacketed glass vessel equippedwith a controlled-speed stirrer. Ports for addition of reactants andwithdrawal of samples as well as nitrogen inlet were provided in thevessel-top assembly. When a stable emulsion of the aqueous dispersedphase in the organic continuous phase was obtained by the action of thestirrer, approximately 0.15 ml of N,N,N',N'-tetramethylenediamine(TEMED) was added to the emulsion and the reaction proceeded at about 0°to 2° C. for another 20 minutes. The resulting suspension ofmicrospheres was poured in 200 ml of cold heptane (0°-5° C.), washedwith heptane, resuspended in ammonium carbonate buffer containing 0.1%of Triton-X-100, washed with pure ammonium carbonate buffer (0.01mole/liter) and freezedried.

                                      TABLE II                                    __________________________________________________________________________    Reaction conditions and characteristics of the product                                 a   b   c   d   e   f   g   h                                        __________________________________________________________________________    Dispersed phase:                                                              Acryloyl-HES                                                                           1.76                                                                              1.76                                                                              1.76                                                                              2.00                                                                              2.00                                                                              2.00                                                                              2.00                                                                              2.00                                     Acrylamide                                                                             0.40                                                                              0.40                                                                              0.40                                                                              0.50                                                                              0.50                                                                              0.50                                                                              0.50                                                                              0.50                                     Alpha-1-PI                                                                             0.34                                                                              0.34                                                                              2.20                                                                              0.60                                                                              0.60                                                                              0.60                                                                              0.60                                                                              0.60                                     Buffer   17.50                                                                             17.50                                                                             15.60                                                                             16.90                                                                             16.90                                                                             16.90                                                                             16.90                                                                             16.90                                    mw.sub.A 3,800                                                                             6,000                                                                             3,800                                                                             3,800                                                                             6,000                                                                             6,000                                                                             6,000                                                                             6,000                                    Continuous phase:                                                             Heptane (ml)                                                                             17                                                                                17                                                                                17                                                                                17                                                                                17                                                                                17                                                                                40                                                                                10                                     Mineral oil (ml)                                                                         43                                                                                43                                                                                43                                                                                43                                                                                43                                                                                43                                                                                20                                                                                50                                     Stirring 1,600                                                                             1,600                                                                             1,600                                                                             1,600                                                                               800                                                                             2,200                                                                             2,200                                                                             2,200                                    (rpm)                                                                         Average diameter                                                                       8.6 14.0                                                                              12.5                                                                              7.6 28.0                                                                              5.8 46.0                                                                              3.6                                      (μm)                                                                       Protein content                                                                        4.7 4.5 22.8                                                                              9.4 --  --  --  --                                       __________________________________________________________________________

EXAMPLE 6

Example 6 was conducted in a manner similar to Example 5, except thatthe product was washed with heptane, then washed with cyclohexane andfinally freeze-dried from cyclohexane. The resulting microspheresexhibited properties analogous to those found in Example 5 but containedessentially all of the protein which had been initially added in thedispersed phase.

EXAMPLE 7

Approximately 1.6 grams of acryloyl-PHEG, prepared according to Example4, 0.58 gram of N-vinyl-2-pyrrolidone and 0.49 gram ofalpha-1-proteinase inhibitor (alpha-1-PI) in 13.5 ml of 0.05 mole/literphosphate buffer pH 7.4 were used as a dispersed phase to preparemicrospheres in a manner similar to that set forth in Example 5d. Theresulting microspheres had an average diameter of 6.7 μm and a proteincontent of 11.2%.

EXAMPLE 8

Approximately 50 mg of microspheres prepared in a manner similar to thatused in Examples 5a-d were suspended in 10 ml 0.05 mole/liter phosphatebuffer pH 7.4 with 0.15 mole/liter NaCl and 0.02% NaN₃. The suspensionswere placed in capped test tubes and were incubated at 37° C. withcontinuous agitation. Samples of the suspensions were withdrawn atconvenient intervals and the microspheres were separated bycentrifugation. The residual amount of the protein in the microspheres,the concentration of protein in microspheres and the concentration ofprotein in the incubation medium were determined using the method ofLowry et al. (O. H. Lowry et al., J. Biol. Chem., 193: 265, 1951). Theamount of alpha-1-PI released as function of time is presented in FIG.2. FIG. 3 describes the cumulative release of alpha-1-PI fromHES-polyacrylamide microspheres in μg of protein per mg of spheres. Theincubation time is plotted in a square root scale. Characteristics ofmicrospheres are those as in Table 2. Characteristics of themicrospheres corresponds to those given in Example 5a-d.

While this invention has been described and illustrated with referenceto certain preferred embodiments thereof, those skilled in the art willappreciate that various changes, modifications and substitutions can bemade therein without departing from the spirit of the invention. It isintended, therefore, that the invention be limited only by the scope ofthe claims which follow.

What is claimed is:
 1. A porous microsphere comprising a biodegradablepolymeric structure having a three-dimensional polymeric network inwhich a biologically active macromolecular agent is physically entrappedtherein and not substantially bonded to the polymeric network, saidpolymeric structure comprising a vinyl derivative of a biodegradablehydrophilic polymer copolymerized with a water-soluble monovinylmonomer, said macromolecular agent able to be released at a controlledrate by diffusion out of the pores and by degradation of the polymericstructure.
 2. The porous microsphere according to claim 1, wherein saidbiodegradable hydrophilic polymer contains at least two vinyl groups orsubstituted vinyl groups per average polymer chain.
 3. The porousmicrosphere of claim 2, wherein the vinyl derivative of thebiodegradable hydrophilic polymer has the formula:

    CH.sub.2 ═CR.sub.1 --(CH.sub.2).sub.n --X              (I)

wherein R₁ represents a hydrogen atom or methyl group; n is 0, 1 or 2; Xrepresents a radical having the formula: ##STR2## and wherein R₂represents the biodegradable hydrophilic polymer which contains at leasttwo vinyl or substituted vinyl groups per average polymer chain.
 4. Theporous microsphere according to claim 3, wherein the biodegradablehydrophilic polymer is selected from the group consisting ofpolysaccharides, proteinaceous polymers, soluble derivatives ofpolysaccharides, soluble derivatives of proteinaceous polymers,polypeptides, polyester and polyorthoesters.
 5. The porous microsphereaccording to claim 4, wherein the biodegradable hydrophilic polymer is apolysaccharide.
 6. The porous microsphere according to claim 5, whereinthe polysaccharide is a starch derivative.
 7. The porous microsphereaccording to claim 4, whereon the biodegradable hydrophilic polymer is apolypeptide.
 8. The porous microsphere according to claim 7, wherein thepolypeptide is poly-(N-hydroxyalkyl)asparagine orpoly-(N-hydroxyalkyl)glutamine.
 9. The porous microsphere according toclaim 1, wherein the water-soluble monovinyl monomer is selected fromthe group consisting of hydrophilic esters and/or amides of acrylic ormethacrylic acids, water-soluble vinyl derivatives, acrylic acid andmethacrylic acid.
 10. The porous microsphere according to claim 1,wherein the ratio between the biodegradable hydrophilic polymer to thewater-soluble monovinyl monomer is in the range of about 1:5 to about40:1 on a weight basis.
 11. The porous microsphere according to claim 1,wherein the biologically active macromolecular agent is a hormone,protein, peptide, vaccine, enzyme or enzyme inhibitor.
 12. The porousmicrosphere according to claim 11, whereon the biologically activemacromolecular agent is a hormone.
 13. The porous microsphere accordingto claim 1, wherein the biologically active macromolecular agent is apeptide.